1. Field of the Invention
This invention relates to novel mutant proteolytic enzymes with improved properties relative to the wild type enzyme in cleaning and detergent formulations, to nucleotide sequences encoding the improved proteases, and to host organisms containing the nucleotide sequences encoding the novel proteases. This invention also includes within its scope new and improved detergent and cleansing compositions containing an effective cleansing amount of said enzymes.
2. Description of the Related Art
Subtilisins are a family of bacterial extracellular proteases with molecular masses of 20,000 to 45,000 daltons produced by a soil bacillus e.g. Bacillus amyloliquefaciens. Proteases are enzymes which catalyze the hydrolysis of peptide linkages in protein and peptide substrates and of ester bonds in some terminal esters. Subtilisins belong to the group of serine proteases which initiate the nucleophilic attack on the peptide (ester) bond by a serine residue at the active site. Subtilisins are physically and chemically well characterized enzymes.
The three-dimensional structure of several subtilisins has been elucidated in detail by X-ray diffraction studies (Betzel, C., Pal, G. P., and Saenger, W. (1988) Eur. J. Biochem. 178, 155-171; Bott, R., Ultsch, M., Kossiakoff, A., Graycar, T., Katz, B., and Power, S. (1988) J. Biol. Chem. 263, 7895-7906; Goddette, D. W., Paech, C., Yang, S. S., Mielenz, J. R., Bystroff, C., Wilke, M., and Fletterick, R. J. (1992) J. Mol. Biol. 228, 580-595; Heinz, D. W., Priestle, J. P., Rahuel, J., Wilson, K. S., and Grutter, M. G. (1991) J. Mol. Biol. 217, 353-371; Kraut, J. (1977) Annu. Rev. Biochem. 46, 331-358; Neidhart, D. J. and Petsko, G. A. (1988) Protein Eng. 2, 271-276; Teplyakov, A. V., Kuranova, I. P., Harutyunyan, E. H., Vainshtein, B. K., Frbommel, C., Hohne, W.-E., and Wilson, K. S. (1990) J. Mol. Biol. 214, 261-279). In spite of this wealth of information the structure/function differences between these closely related subtilisins have not been explained.
Subtilisins are widely used in commercial products (for example, in laundry and dish washing detergents, contact lens cleaners) and for research purposes (catalysts in synthetic organic chemistry). One member of the subtilisin family, a highly alkaline protease for use in detergent formulations has been described in patent application WO 91/02792. This Bacillus lentus alkaline protease (BLAP) can be obtained in commercial quantities from Bacillus licheniformis ATCC 53926 strain transformed by an expression plasmid harboring the wild type BLAP gene under the control of the B. licheniformis ATCC 53926 alkaline protease gene promoter. The crystal structure of BLAP has been deduced (Goddette, D. W., et al. (1992) J. Mol. Biol. 228, 580-595; WO 92/21760), and the coordinates have been deposited with the Brookhaven Protein Data Bank.
Unless other wise noted the numbering of the amino acid positions is according to the sequence in BLAP (269 amino acids), which differs from that of subtilisin BPN' (275 amino acids). When aligned for optimal sequence homology the following pattern emerges. In BLAP positions 1 to 35, 36 to 54, 55 to 160, and 161 to 269 correspond to positions 1 to 35, 37 to 55, 57 to 162, and 167 to 275, respectively, in subtilisin BPN'.
In order to describe protease variants according to the invention, the following nomenclature is used: [Original Amino Acid; Position from N-terminus of the mature enzyme; Substituted Amino Acid]. For example, the substitution of valine with isoleucine at position 4 in BLAP is designated as V4I. The list of abbreviations for amino acids is shown in Table 1.
TABLE 1 Amino Acid Nomenclature A = Ala = Alanine C = Cys = Cysteine D = Asp = Aspartic acid E = Glu = Glutamic acid F = Phe = Phenylalanine G = Gly = Glycine H = His = Histidine I = Ile = Isoleucine K = Lys = Lysine L = Leu = Leucine M = Met = Methionine N = Asn = Asparagine P = Pro = Proline Q = Gln = Glutamine R = Arg = Arginine S = Ser = Serine T = Thr = Threonine V = Val = Valine W = Trp = Tryptophan Y = Tyr = Tyrosine
Multiple mutations within the same protein molecule are indicated as the sum of individual mutations, i.e. S3T+V4I+A188P+V193M+V199I.
Protection against thermal and chemical inactivation and improvement of washing and cleaning performance are primary objectives in research and development of proteases for technical as well as for commercial applications. A large number of enzymes including subtilisins have been generated by random and site-specific mutagenesis and they provide some guidelines for a rational approach to the improvement of thermal and chemical stability (Estell, D. A., Graycar, T. P., and Wells, J. A. (1985) J. Biol. Chem. 260, 6518-6521; Matsumura, M., Becktel, W. J., Levitt, M., and Matthews, B. W. (1989) Proc. Natl. Acad. Sci. USA 86, 6562-6566; Pantoliano, M. W., Whitlow, M., Wood, J. F., Rollence, M. L., Finzel, B. C., Gilliland, G. L., Poulos, T. L., and Bryan, P. N. (1988) Biochemistry 27, 8311-8317; Russell, A. J. and Fersht, A. R. (1987) Nature 328, 496-500; Siezen, R. J., De Vos, W. M., Leunissen, J. A. M., and Dijkstra, B. W. (1991) Protein Eng. 4, 719-737; van Ee, J. H. (1991) Chimicaoggi (7/8), 31-35; Wells, J. A. and Estell, D. A. (1988) Trends Biochem. Sci. 13, 291-297). The modulation of enzymic activity, in particular rate enhancement or optimization for a subset of substrates, is a far more complex problem. EP 0260105 teaches the construction of subtilisin BPN' mutants with altered transesterification rate/hydrolysis rate ratios and nucleophile specificities by changing specific amino acid residues within 15 .ANG. of the catalytic triad. Russell, A. J., and Fersht, A. R. (1987) J. Mol. Biol. 193: 803-813, teach the isolation of a subtilisin BPN' mutant (D099S) that had a change in the surface charge 14 to 15 .ANG. from the active site. This substitution causes an effect on the pH dependence of the subtilisin's catalytic reaction. Neither of these publications teach how to predict amino acid alterations that will improve the wash performance of the protease. EP 0130756, EP 0247647, and U.S. Pat. No. 4,760,025 teach a saturation mutation method where one or multiple mutations are introduced into the subtilisin BPN' at amino acid residues (BPN' numbering) Asp32, Asn155, Tyr104, Met222, Gly166, His64, Ser221, Gly169, Glu156, Ser33, Phe189, Tyr217, and/or Ala152. Using this approach mutant proteases exhibiting improved oxidative stability, altered substrate specificity, and/or altered pH activity are obtained. These publications also teach that mutations within the active site region of the protease are the most likely to influence activity. However, neither EP0130756, EP 0247647, nor U.S. Pat. No. 4,760,025 teach a method for predicting amino acid alterations that will improve the wash performance of the protease.
Most of the information on the catalytic activity of subtilisins has been collected by examining the hydrolysis of small, well defined peptide substrates. Yet, little is known about interactions with large protein substrates. This is especially true for the wash performance of proteases where the substrate is attached to a textile surface and catalysis takes place in presence of interfering compounds such as bleach, tensides, and builders.
EP 0328229 teaches the isolation and characterization of PB92 subtilisin mutants with improved properties for laundry detergent applications based upon wash test results. It teaches that biochemical properties are not reliable parameters for predicting enzyme performance in the wash. Methods for selection of mutations involve the substitution of amino acids by other amino acids in the same category (polar, nonpolar, aromatic, charged, aliphatic, and neutral), the substitution of polar amino acids asparagine and glutamine by charged amino acids, and increasing the anionic character of the protease at sites not involved with the active site. No method for identifying which specific amino acids should be altered is taught. Patent application WO 91/00345 (Novo-Nordisk) teaches a method to improve the wash performance of a subtilisin by the modification of the isoelectric point of subtilisin Carlsberg and subtilisin 309 to match the pH of the washing solution, where the enzyme is supposed to be used. However, since the highly alkaline subtilisins have pI values close to the pH of detergents under European washing conditions, this approach offers no obvious advantage. WO 91/00345 also teaches that changes in amino acids more than 15 .ANG. from the catalytic triad can result in changes in the kinetic properties of the enzyme. A total of 116 different amino acids out of a total of 269 amino acids present in subtilisin 309 are suggested as possible sites for substitution, addition or deletion to modify the net electric charge of the enzyme.
Patent application WO 92/11357 (Novo-Nordisk) teaches increased pH-stability and improved washability of subtilisins by reduction of pH-dependent charges of the molecule. This means the introduction of mutations to approach substantial constancy of charge over a pH range.
Preferably, an almost zero net charge change in the pH range from 7 to 11. European patent application No. 0 57 049 A1 discloses certain mutant proteolytic enzymes. These enzymes are said to have at least 70% homology with the amino acid sequence of PB92 serine protease and differ by at least one amino acid corresponding to 99, 102, 116, 126, 127, 128, 130, 160, 203, 211, and 212 in the PB92 serine protease. The mutant protease is prepared by growing a microorganism host strain transformed with an expression vector comprising a DNA sequence and encoding a mutant protease to produce the designed mutant protease.